Methods and compositions for regulating imidazoline receptors

ABSTRACT

The present invention relates to methods and compositions for regulating the activity of imidazoline receptors. In particular, the invention relates to pharmaceutical compositions comprising imidazoleacetic acid-ribotide (IAA-RP), imidazoleacetic acid-riboside (IAA-R) and its related congeners such as pros-linked ribotide and riboside. The invention is based on the discovery that IAA-RP and to a lesser extent IAA-P bind with a high affinity to imidazoline receptors. Antibodies to IAA-RP, IAA-R are additionally provided, as well as screening methods for identification of compounds that either promote or antagonize the activity of IAA-RP, IAA-R and its related congeners. The invention further relates to diagnostic and prognostic methods for detection of abnormalities in levels or activity of IAA-RP and IAA-R. The invention encompasses treatment of disorders related to the imidazoline system, including hypertension, glaucoma, psychiatric (e.g., depression), neurological (e.g., motor disorders, neurodegenerative disorders), diabetes and disorders related to platelet aggregation.

The present invention relates to methods and compositions for regulatingthe activity of imidazoline receptors. In particular, the inventionrelates to pharmaceutical compositions comprising imidazoleaceticacid-ribotide (IAA-RP) and imidazoleacetic acid-riboside (IAA-R). Theinvention is based on the discovery that IAA-RP and to a lesser extentIAA-R bind with a high affinity to imidazoline receptors. Antibodies toIAA-RP and IAA-R are additionally provided, as well as screening methodsfor identification of compounds that either promote or antagonize theactivity of IAA-RP and IAA-R. The invention further relates todiagnostic and prognostic methods for detection of abnormalities inlevels or activity of IAA-RP and IAA-R. The invention encompassestreatment of disorders related to the imidazoline system, includinghypertension, glaucoma, psychiatric (e.g., depression), neurological(e.g., motor disorders, neurodegenerative disorders), diabetes anddisorders related to platelet aggregation.

BACKGROUND OF THE INVENTION

Imidazoline receptors are now generally recognized as a unique set ofnon-adrenergic high affinity binding sites for a number of agents thatto date also bind to α₂-adrenergic receptors (Eglen, R. M. et al., 1998,Trends in Pharmacol. Sci. 19: 381-390; Regunathan, S. and Reis, D. J.,1996, Ann. Rev. Pharmacol. Toxicol. 36:511-44). Although membrane-boundimidazoline receptors have not yet been cloned, evidence includingdifferences in selectivity and binding affinity of ligands, thestructure of binding proteins and cellular distribution indicate thatthey are different from α₂-adrenergic receptors. The nonadrenergicimidazoline receptors are important in mediating the hypotensive actionsof clinically important imidazoline drugs such as clonidine, rilmenidineand moxonidine.

For example, unique imidazoline receptors, are present in pancreaticislet and beta-cells (Morgan, N. G., et al., 1995, Ann. N.Y. Acad. Sci.763: 361-373). Activation of these receptors by imidazolines causesrelease of insulin. Much of this activity is due to imidazoline-inducedclosure of K⁺ channels such as the K⁺ ATP-sensitive channels whichpermits intracellular levels of K+ to accumulate, causing celldepolarization and eventual exocytosis of hormone or transmitter intoplasma or extracellular fluid. It is noteworthy that channels such asthe K⁺ ATP-sensitive channels exist throughout the body, and areparticularly abundant in brain. These pancreatic imidazoline receptorshave recently been designated as I₃ receptor subtypes (Eglen, R. M. etal., 1998, TIPS 19: 381-390).

Chan et al. (1997, Brit. J. Pharmacol. 120: 926-932), showed thatimidazolines and preparations of CDS (clonidine-displacing substance,see below) from bovine brain caused release of insulin and stimulated K⁺ATP channels.

One or more endogenous ligands selectively bind to the imidazolinereceptors although attempts to identify this endogenous ligand(s) hasfailed. A possible ligand, referred to as “clonidine displacingsubstance” (CDS), has been discovered as an entity isolated frommammalian brain and the periphery that is capable of displacingradio-labeled clonidine and its radio-labeled congeners from membranes(Atlas, L. et al., 1987, J. Cardiovascular Pharmacology 10(Suppl. 12):S122-S127; Atlas, D. 1991, Biochemical Pharmacology 41: 1541-1549;Atlas, D., 1995, Annals of the New York Academy of Sciences763:314-324). Antibodies have been prepared against the drug clonidine,which presumably interact with CDS. Such antibodies are found to beimmunoreactive in tissues throughout the body and also show aheterogeneous regional distribution within the brain.

A recent study proposed that agmatine, a known compound isolated frombovine brain, is CDS (Li, G. et al., 1994, Science 263:966-968;Regunathan, S. and Reis, D. J., 1996, Ann. Rev. Pharmacol. Toxicol. 36:511-544; but see Eglen, R. M. et al., 1998, TIPS 19: 381-390). Agmatinewas further suggested to be an endogenous neurotransmitter because itwas found within an extract of CDS activity from whole brain and becauseit appeared to bind to a class of imidazoline receptors. However,comparisons of the biological activities of agmatine, e.g., effects onblood pressure versus effects of endogenous clonidine-displacingsubstance at imidazoline and α₂-adrenergic receptors produced invirtually all laboratories indicated that agmatine differed from“classical CDS.” For example, agmatine displaces labeled clonidine froma subset of its nonadrenergic binding sites identified as imidazoline2_(A) and 2_(B) sites. However, because those I_(2A) and I_(2B) sitesare now known to be enzymes, i.e. portions of monoamine oxidase A and B,the search for the identity of CDS that acts at membrane-boundimidazoline receptors has continued (Eglen, R. M. et al., 1998, TIPS 19:381-390).

Several laboratories have harvested CDS and most preparations showsimilar physiochemical properties. There is widespread consensus thatCDS is present in small amounts in the brain, cerebrospinal fluid andperiphery (including plasma) of mammals. It is soluble in water andmethanol, but generally insoluble in organic solvents. Size exclusionchromatography indicated that it is a small molecule (≦1000 Da). CDS isresistant to several proteases, including trypsin and chymotrypsin, andis devoid of amino acids; thus it is not a peptide. CDS appears to haveno free amino groups as activity is retained following reaction withfluorescamine and ninhydrin. CDS is stable in both weak acids (pH 2) andweak bases (pH 10.5), is thermostable (at 110° C.) and retains activityfollowing multiple freeze-thaw and lyophilization cycles. Because CDScan be retained on both anion and cation exchange resins and because itsmigration patterns shifted markedly with changes in ambient pH on gelelectrophoresis, it is very likely that CDS is amphoteric, possibly azwitterion. In addition, CDS shows maximal UV absorbance between 206-220nm.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for regulatingthe activity of imidazoline receptors. Specifically, the inventionrelates to compositions comprising imidazoleacetic acid-ribotide(IAA-RP) which binds with high affinity to at least 2 subsets ofimidazoline receptors, imidazoleacetic acid-riboside (IAA-R) which bindswith a slightly lower affinity, and to its related congeners. Asdemonstrated herein, IAA-RP binds to imidazoline receptors and in onecase stimulates well defined receptor-mediated signal transductionevents such as release of arachidonic acid. Further, the release ofarachidonic acid, an imidazoline I₁-receptor mediated event, isinhibited in the presence of the imidazoline I₁ receptor antagonistefaroxan. The discovery that IAA-RP and IAA-R bind to imidazolinereceptors provides new targets for therapeutic methods aimed atamelioration of imidazoline system related disorders.

The present invention includes pharmaceutical compositions comprisingIAA-RP, IAA-R, derivatives and analogs thereof, which can be utilized toregulate the activity of imidazoline and imidazoline-like receptors.Such compositions can be utilized to treat disorders related to theimidazoline system such as hypertension, glaucoma, psychiatric (e.g.depression), neurological (e.g. motor disorders, neurodegenerativedisorders), diabetes and disorders involving platelet aggregation.

The invention further provides for antibodies to IAA-RP and IAA-R. Suchantibodies can be utilized to ameliorate symptoms associated withimidazoline system-related disorders. For example, in the case of ananti-IAA-RP antibody, such an antibody would specifically bind to IAA-RPand possibly disrupt the ability of IAA-RP to bind to imidazolinereceptors thereby preventing receptor mediated signal transductionevents. Additionally, anti-IAA-RP and anti-IAA-R antibodies can be usedas diagnostic and prognostic indicators of imidazoline system relateddisorders. For example, diagnostic methods can be utilized to detectabnormalities in the levels or tissue distribution of IAA-RP and/orIAA-R relative to normal levels. The antibodies of the invention canalso be used in screening methods for detection of a predisposition toimidazoline system based disorders in an individual.

The invention further relates to methods for identification of compoundswhich promote or antagonize signal transduction events stimulated by thebinding of IAA-RP or IAA-R to imidazoline receptors. Such compounds canact as therapeutic agents in the amelioration of a wide range ofimidazoline system based disorders. The invention further relates tomethods for identification of compounds that regulate the synthesis, ordegradation of IAA-RP or IAA-R.

Finally, the invention relates to treatment of imidazoline baseddisorders, such as for example, hypertension, glaucoma, psychiatric(e.g. depression), neurological (e.g. motor disorders, neurodegenerativedisorders), diabetes and disorders involving platelet aggregation byadministering compositions comprising IAA-RP, IAA-R, or compounds thatpromote or antagonize IAA-RP or IAA-R activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical structures of IAA-RP and IAA-R.

FIG. 2. IAA-RP inhibition curves after selective blockade of eitherα₂-adrenergic or imidazoline receptor sites in the RVLM (Rostal Ventrallateral Medulla) of brain. Data represent the mean±S.E.M. of 4 to 6experiments, each conducted in triplicate. VLM membranes were incubatedwith increasing concentrations of IAA-RP in the presence of vehiclealone (1.0 mM acetic acid; control), with epinephrine (0.1 mM) added tomask α₂-receptors, or with cimetidine (10 μM) added to mask imidazolinereceptors. Each curve was normalized to the total specific binding underthat condition. Results of curve-fitting analysis are shown in Table 2.

FIG. 3. Effect of phosphatase treatment on the dose-dependent inhibitionof ¹²⁵I-iodoclonidine binding to adrenomedullary cell membranes by brainextract containing CDS.

FIG. 4. Stimulation of arachidonic acid release by IAA-RP, animidazoline I₁response, and inhibition of arachidonic acid release byefaroxan an I₁ antagonist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions comprisingimidazoleacetic acid-ribotide (IAA-RP), imidazoleacetic acid-riboside(IAA-R) and their related congeners for use in regulating the activitiesof imidazoline receptors. The invention is based on the discovery thatIAA-RP and to a lesser extent, IAA-R have significant affinities forimidazoline receptors. Further, binding of IAA-RP to the imidazoline I₁receptor in the adrenal medulla stimulates release of arachidonic acid(AA), an imidazoline I₁ receptor-mediated signal transduction response.The release of arachidonic acid is inhibited in the presence ofefaroxan, a known antagonist of the imidazoline I₁ receptor.

The invention encompasses compositions comprising IAA-RP, IAA-R and/orrelated congeners which can be utilized to regulate activities ofimidazoline receptors. The invention further provides for antibodies toIAA-RP and IAA-R. Such antibodies can be utilized to ameliorateimidazoline related disorders. Alternatively, anti-IAA-RP and anti-IAA-Rantibodies can be used diagnostically and prognostically to detectabnormalities in levels or tissue distribution of IAA-RP and/or IAA-Rrelative to normal levels.

The discovery that IAA-RP, IAA-R and related congeners bind toimidazoline receptors provides a new target for therapeutic methodsaimed at amelioration of imidazoline system related disorders. Thus, theinvention further relates to methods for identification of compoundswhich promote or antagonize the biological activity stimulated byIAA-RP, IAA-R and related congeners that bind to imidazoline receptors.Such compounds can act as therapeutic agents in the amelioration of awide range of imidazoline based disorders.

Finally, the invention relates to treatment of imidazoline system baseddisorders, such as hypertension, glaucoma, psychiatric (e.g.depression), neurological (e.g. motor disorders, neurodegenerativedisorders), diabetes and disorders involving platelet aggregation byadministering compositions comprising IAA-RP, IAA-R, related congeners,and/or compounds that promote or antagonize IAA-RP or IAA-R activity.

5.1. Pharmaceutical Compositions Containing IAA-RP. IAA-RP and theirRelated Congeners

The present invention provides pharmaceutical compositions containingimidazoleacetic acid ribotide (IAA-RP) or imidazoleacetic acid riboside(IAA-R). FIG. 1 depicts the tele-linked isomers of IAA-RP and IAA-R.IAA-RP, or tele-linked IAA-RP, is also known as imidazole-4-aceticacid-ribotide as well as 1-(β-D-ribofuranosyl)-imidazole-4-acetic acid5′ phosphate. Its metabolite, IAA-R or tele-linked IAA-R, is also knownas imidazole-4-acetic acid-riboside or as1-(β-D-ribofuranosyl)-imidazole-4-acetic acid. Both compounds exhibitcovalent imidazole-furan linkage with the imidazole nitrogen atomfurthest from the methylene-carboxy side chain. This atom is termed thetele-N or N^(τ), analogous to the IUPAC terms used to define thenitrogen atoms of histidine. The corresponding pros-linked isomers ofIAA-RP and IAA-R, are termed 1-(β-D-ribofuranosyl)-imidazole-5-aceticacid 5′ phosphate or pros-linked IAA-RP and1-(β-D-ribofuranosyl)-imidazole-5-acetic acid or pros-linked IAA-R,respectively. For these compounds, the imidazole-furan linkage is withthe pros-N or N^(π), i.e., the imidazole ring nitrogen closest to themethylene-carboxy side chain.

In addition, pharmaceutical compositions comprising congeners andderivatives of IAA-RP and IAA-R which have a high affinity forimidazoline receptors are within the scope of the present invention. Asreferred to herein, congeners are defined as chemical compounds closelyrelated to another in structure and exerting similar or antagonisticeffects. For example, a structural isomer of IAA-RP with, for example,the addition of one or more phosphate or phosphonate groups. In general,the invention comprises ribosylated imidazoles, such asimidazole-furanosyl ribotides and ribosides, including but not limitedto compounds such as 5-amino-4-imidazole carboxamide-ribosephosphate(AICARP, also commonly abbreviated ZMP), an intermediate in the de novosynthesis pathway of purine nucleotides. In addition, substitution of amethylene group (—CH₂—) for the oxygen atom that links the 5′ carbon tothe phosphate atom in IAA-RP, can be done to produce a molecule that ismuch more resistant to enzymatic dephosphorylation by phosphatases or 5′nucleases. Such molecules would have more desirable pharmacokineticproperties. In addition, 2′ or 3′ deoxy-IAA-RP which retain affinity forthe imidazoline receptor are within the scope of the present invention.Compounds within the scope of the invention also include esters ofIAA-RP, such as carboxy-methyl or carboxy-ethyl esters of IAA-RP. Suchcompounds are more lipid soluble, and thus, would diffuse more rapidlyacross biological barriers such as the blood-brain barrier or cellslining the gut.

Additionally, molecules within the scope of the invention include thosecompounds with linkage of the furan to the number 2 carbon atom of theimidazole ring, i.e, the carbon atom in between the two imidazole ringnitrogens. Alternatively, the furan ring may be linked to the number 2carbon atom of the imidazole ring, with reduction of the double bondbetween carbons 4 and 5, leading to the conversion of the imidazole ringto an imidazoline ring or an imidazoline-like ring.

Methods for synthesis of IAA-RP and IAA-R are well known to those ofskill in the art and include both biosynthetic and organic methods ofsynthesis. Methods for recovery and purification of IAA-RP and IAA-Rfrom biological samples are described in various references (Karjala, S.A., 1955, J. Amer. Chem. Soc. 77:504-505; Tabor, H. and Hayaishi, O.,1955, J. Amer. Chem. Soc. 77:505-506: Crowley, G. M., 1964, J. Biol.Chem. 239: 2593-2601; Karjala, S. A. et al., 1956, J. Biol. Chem.0.219:9-12; Beaven, M. A. et al., 1974, Europ. J. Pharmacol. 29:138-146;Moss, J. et al., 1976, J. Clin. Invest. 58:137-141; Robinson, J. D. andGreen, J. P., 1964, Nature 203:1178-1179; Beaven, M. A. et al., 1976,Experientia 32:1180-1182; Thomas, B. and Prell, G. D., 1993, Soc.Neurosci. Abst. 19:85; Thomas, B. and Prell, G. D., 1995, J. Neurochem.65: 818-826; Thomas, B. et al., 1995, Soc. Neurosci. Abst. 21: 1857).

In addition, organic synthesis of IAA-R can be carried out using themethod of Bauer (1958, BBA 30:219; and 1962, J. Org. Chem. 27:167-170;Baddiley, J. et al, 1958, J. Chem. Soc. 3743-3745). The 5′ hydroxylgroup on IAA-R can be phosphorylated as presented in Matulic′-Adamic′,J. and Watanabe, K. A. (1991, Korean J. Med. Chem. 1:54-64) to yieldIAA-RP. In addition, IAA-RP can be enzymatically synthesized from IAA-Rusing for example, enzymes such as adenosine kinase (ATP:adenosine 5′phosphotransferase) to transfer a terminal phosphate from ATP to IAA-R,to produce IAA-RP. The resultant IAA-RP can be rapidly purified usingany of a variety of methods including anion exchange, HPLC and TLC. Someother congeners of tele-linked IAA-RP and IAA-R (including synthesis ofpros-linked IAA-RP and IAA-R) (FIG. 1) are described elsewhere (e.g.Matulic′-Adamic′, J. and Watanabe, K. A., 1991, Korean J. Med. Chem. 1:54-64).

In some instances it may be advantageous to transfer a labeled terminalphosphate from ATP to IAA-R to produce labeled IAA-RP, i.e., IAA-R³² P.Such labeled IAA-RP will have a number of different uses including usein binding and receptor studies, during screens developed foridentification of compounds having an affinity for imidazolinereceptors, for isolation of imidazoline receptors as well as foranalysis of the binding domains of imidazoline receptors. In addition,labeled IAA-RP can be used in pulse-chase studies of IAA-RP metabolism,analysis of IAA-RP's pharmacokinetic properties, and analysis of IAA-RPrecovery in analytical methods.

To determine whether the tele- or pros- form of IAA-RP and IAA-R ispresent in the brain, pulse-chase experiments involving theadministration of radiolabeled precursor produce IAA-RP and IAA-R weredone. Using an anion-exchange HPLC/UV method we were able to separateimidazole-4-acetic acid-ribotide from imidazole-5-acetic acid-ribotide;it was determined that IAA-RP is present in rat brain, human brain,cerebrospinal fluid and preparations of CDS harvested from bovinebrains. Only imidazole-4-acetic acid-ribotide, i.e., the isomer in whichthe furan ring is linked to the imidazole tele-nitrogen (the nitrogenlocated furthest from the CH₂COOH side chain; see, FIG. 1) wasconsistently observed. Furthermore, HPLC analysis of biological samplesmixed with authentic tele-linked IAA-RP prepared by organic synthesis,produced a larger UV absorption peak coincident with the tele-linkedIAA-RP retention time. No split peaks or additional peaks were observedin this region of the HPLC chromatogram.

In contrast, mixing parallel biological aliquots with authenticimidazole-5-acetic acid-ribotide, i.e., the isomer in which the furanring is linked to the pros or π ring nitrogen (the imidazole ringnitrogen located closest to the —CH₂COOH side chain) produced a novelnonphysiological peak where previously there had been essentiallybaseline absorbance. This new peak was approximately 2 minutes behindthe endogenous IAA-RP peak, a shift in retention virtually identical tothat observed for I-5-AA-RP when authentic I-4-AA-RP and I-5-AA-RP wereanalyzed alone or together. Thus, there appears to be little or noI-5-AA-RP in the biological material we analyzed. These observations areconsistent with observations made using gas chromatography-massspectrometry where the tele-linked IAA-riboside, but not pros-linkedIAA-riboside, was present in biological samples including rat brain. Inaddition, only the tele-linked isomer of IAA-RP shows significantactivity for displacing clonidine from its nonadrenergic membranebinding sites in the adrenal medulla. The pros-linked IAA-RP isomer wasdevoid of this activity.

The fact that pros-linked IAA-RP or pros-linked IAA-R does not seem tobe present in samples of rat brain nevertheless suggest a number of usesof pros-linked IAA-RP. For example, pros-linked IAA-RP, or pros-linkedIAA-R, can be used as an internal standard in analytical techniques suchas for example, in HPLC methods, to determine recovery of endogenoustele-linked IAA-RP. The pros-linked IAA-RP can also be used as a controlsubstance when the activities of the endogenous tele-linked IAA-RP areto be assessed. More importantly, in cases such as with imidazoline I₃or I₃-like receptors, pros-linked IAA-R has its own activities oninsulin release and interactions with potassium channels.

The pharmaceutical compositions of the invention comprise an effectiveamount of IAA-RP, IAA-R, or related congeners and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such pharmaceutical compositions will contain a therapeuticallyeffective amount of IAA-RP, IAA-R, and/or a related congener, preferablyin purified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

5.2 Generation of Antibodies to IAA-RP, IAA-R or Related Congeners

According to the invention, IAA-RP, IAA-R or related congeners may beused as immunogens to generate antibodies which immunospecifically bindsuch immunogens. Such antibodies include, but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fabexpression library.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to IAA-RP or IAA-R or derivatives or analogsthereof. In a particular embodiment, rabbit polyclonal antibodies to anepitope of IAA-RP or IAA-R can be obtained. For the production ofantibody, various host animals can be immunized by injection with IAA-RPor IAA-R, or a synthetic version, or derivative thereof, including butnot limited to rabbits, mice, rats, etc. Various adjuvants may be usedto increase the immunological response, depending on the host species,and including, but not limited to Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum.

For preparation of monoclonal antibodies directed toward IAA-RP or IAA-Ror analogs thereof, any technique which provides for the production ofantibody molecules by continuous cell lines in culture may be used. Forexample, the hybridoma technique originally developed by Kohler andMilstein (1975, Nature 256:495-497), as well as the trioma technique,the human B-cell hybridoma technique (Kozbor et al., 1983, ImmunologyToday 4:72), and the EBV hybridoma technique to produce human monoclonalantibodies (Cole et al., 1985, in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96). According to the invention,human antibodies may be used and can be obtained by using humanhybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:2026-2030) or by transforming human B cells with EBV virus in vitro(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, pp. 77-96). In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al.,1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al.,1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) canbe used; such antibodies are within the scope of this invention.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). Various immunoassays known in theart can be used to determine the binding characteristics of theantibodies, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, insitu immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), IAA-RP or IAA-R precipitation reactions, agglutinationassays (e.g., gel agglutination assays, hemagglutination assays),complement fixation assays, immunofluorescence assays, protein A assays,and immunoelectrophoresis assays, etc. In one embodiment, antibodybinding is detected by detecting a label on the primary antibody. Inanother embodiment, the primary antibody is detected by detectingbinding of a secondary antibody or reagent to the primary antibody. In afurther embodiment, the secondary antibody is labeled. Many means areknown in the art for detecting binding in an immunoassay and are withinthe scope of the present invention.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity, e.g., for imaging IAA-RP andIAA-R molecules, measuring levels thereof in appropriate physiologicalsamples, in diagnostic methods, etc. In another embodiment of theinvention, anti-IAA-RP or anti-IAA-R antibodies and fragments thereofcontaining the binding domain can be used to regulate the activity ofimidazoline receptors.

5.3. Diagnosis and Screening

Anti-IAA-RP and -IAA-R antibodies, have uses in diagnostics. Suchmolecules can be used in assays, such as immunoassays, to detect,prognose, diagnose, or monitor various conditions, diseases, anddisorders affecting the imidazoline system. In particular, such animmunoassay is carried out by a method comprising contacting a samplederived from a patient with an anti-IAA-RP or anti-IAA-R antibody underconditions such that immunospecific binding can occur, and detecting ormeasuring the amount of any immunospecific binding by the antibody. In aspecific aspect, such binding of antibody, in samples derived from thepatient, can be used to detect aberrant IAA-RP or IAA-R localization oraberrant (e.g., high, low or absent) levels of IAA-RP or IAA-R. In aspecific embodiment, antibody to IAA-RP or IAA-R can be used to assay ina patient tissue or serum sample for the presence of IAA-RP or IAA-Rwhere an aberrant level of IAA-RP or IAA-R is an indication of adiseased condition. By “aberrant levels,” is meant increased ordecreased levels relative to that present, or a standard levelrepresenting that present, in an analogous sample from a portion of thebody or from a subject not having the disorder.

The immunoassays which can be used, include but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew.

Kits for diagnostic use are also provided that comprise in one or morecontainers an anti-IAA-RP or IAA-R antibody, and, optionally, a labeledbinding partner to the antibody. Alternatively, the anti-IAA-RP or IAA-Rantibody can be labeled (with a detectable marker, e.g., achemiluminescent, enzymatic, fluorescent, or radioactive moiety).

5.4. Screening for Agonists and Antagonists of IAA-RP, IAA-R and RelatedCongeners

A variety of different assay systems can be designed and used toidentify compounds or compositions that modulate IAA-RP or IAA-Ractivity, and therefore, may be useful to regulate imidazoline receptorsand useful in the treatment of diseases associated with the imidazolinesystem.

In accordance with the invention, cell-based assay systems are used toscreen for compounds that modulate the activity of IAA-RP and IAA-R andthereby modulate the activity of imidazoline receptors. Compounds thatmay affect IAA-RP and/or IAA-R activity include, but are not limited tocompounds that promote (agonists) or block (antagonists) activation ofimidazoline receptors.

To this end, cells that endogenously express imidazoline receptors canbe used to screen for compounds that modulate IAA-RP or IAA-R activity.Cells that express imidazoline receptors can be further engineered toincorporate a reporter molecule, the expression of which is linked tothe signal transduced by IAA-RP or IAA-R or related congener activationof imidazoline receptors to aid in identification of compounds thatmodulate activity. Cells to be used to screen for compounds are cellsthat respond to activation of imidazoline receptors by IAA-RP, or IAA-R,or their congeners, e.g., as measured by a chemical, physiological,biological, or phenotypic change. For example, a test compound may beused to assess the ability for IAA-RP, IAA-R or a related congener tobind to imidazoline receptors and thereby inhibit or activate signaltransduction processes. In one case, release of arachidonic acid can beused to assess imidazoline I₁ activity. In addition, release of insulinand/or closure of K⁺ ATP channels can be used to assess imidazoline I₃activity.

In utilizing such cell-based assay systems, cells that expressimidazoline receptors are exposed to a test compound or to vehiclecontrols (e.g., placebos). After exposure, the cells can be assayed tomeasure the expression and/or activity of components of the signaltransduction pathway affected by IAA-RP, IAA-R or their congeners. Forexample, in cells of the adrenal medulla that express imidazoline I₁receptors, binding of IAA-RP and IAA-R are associated with release ofarachidonic acid; thus, in a specific embodiment of the invention,assays may be designed to measure arachidonic acid. The ability of atest compound to decrease levels of arachidonic acid release, ascompared to those levels seen with cells treated with a vehicle control,indicates that the test compound inhibits signal transduction mediatedby binding of IAA-RP, IAA-R or a related congener to an imidazoline I₁receptor. In addition, assays may be developed to measure IAA-RP orIAA-R induced release of insulin from pancreatic β-cells or release ofcatecholamines from chromaffin cells.

Non-cell based assays may be used to identify compounds that bind toIAA-RP or IAA-R molecules. The principle of assays used to identifycompounds that bind to IAA-RP or IAA-R involves preparing a reactionmixture of the molecules and the test compound under conditions and fortime sufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The identity of the bound test compound is then determined.

The screening assays are accomplished by any of a variety of commonlyknown methods. For example, one method to conduct such an assay involvesanchoring the IAA-RP, IAA-R, or a related congener onto a solid phaseand detecting IAA-RP or IAA-R/test compound complexes retained on thesolid phase at the end of the reaction. In one embodiment of such amethod, the IAA-RP or IAA-R reactant is anchored onto a solid surface,and the test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

In practice, microtitre plates can be utilized conveniently as the solidphase. The anchored component is immobilized by non-covalent or covalentattachments. The surfaces may be prepared in advance and stored. Inorder to conduct the assay, the non-immobilized component is added tothe coated surfaces containing the anchored component. After thereaction is completed, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the solidsurface, e.g., using a labeled antibody specific for the previouslynon-immobilized component.

Alternatively, a reaction is conducted in a liquid phase, the reactionproducts separated from unreacted components using an immobilizedantibody specific for IAA-RP, IAA-R, or a related congener, fusionprotein or the test compound, and complexes detected using a labeledantibody specific for the other component of the possible complex todetect anchored complexes.

Assay for compounds that interfere with the interaction of IAA-RP,IAA-R, or related congeners with imidazoline receptors can be performed.Ligand/receptor interactions can be detected at the end of the reactioncomparing interactions in the presence or absence of test compound. Theorder of addition of test compounds can be varied to obtain differentinformation about the compounds being tested. For example, testcompounds that interfere with the interaction by competition can beidentified by conducting the reaction in the presence of the testcompound, i.e., by adding the test compounds to the reaction mixtureprior to or simultaneously with IAA-RP, IAA-R, or a related congener.Alternatively, test compounds that disrupt preformed complexes, i.e,those compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction after the complexes have formed.

In addition, assays may be used to identify compounds that interferewith the expression or degradation of IAA-RP or IAA-R. For example,IAA-RP is synthesized by imidazoleacetic acid phosphoribosyltransferase(IPRT) (Thomas, B. and Prell, G. D., 1995, J. Neurochem. 65:818-826). Inan embodiment of the present invention, assays may be developed toidentify compounds which inhibit IPRT thereby reducing levels of IAA-RPand/or IAA-R. Alternatively, such assays may be used to identifycompounds that activate IPRT resulting in elevated levels of IAA-RPand/or IAA-R. In a non-limiting embodiment of the invention, antibodiesdirected against IAA-RP or IAA-R may be used in the assays of theinvention to detect changes in levels of IAA-RP and/or IAA-R.

IAA-RP is metabolized to IAA-R by dephosphorylation through the actionof either 5′ phosphatases and/or 5′ nucleotidases (endo or ecto).Inhibition of such phosphatases or nucleotidases would result inaccumulation of IAA-RP and/or reductions in levels of IAA-RP.Alternatively, activation of such phosphatases and/or nucleotidaseswould result in reduction in levels of IAA-RP and/or an increase inlevels of IAA-R. In yet another embodiment of the invention, assays maybe developed to identify compounds capable of regulating the activity of5′ phosphatases and/or 5′ nucleotidases.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to inorganic compounds, peptides,antibodies and fragments thereof, and other organic compounds (e.g.,peptidomimetics) that bind to imidazoline receptors and either mimic theactivity of IAA-RP, IAA-R or a related congener (i.e., agonists) orinhibit the activity of IAA-RP, IAA-R (i.e., antagonists). Compounds mayinclude, but are not limited to, peptides such as, for example, solublepeptides, including but not limited to members of random peptidelibraries; (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghten, R. et al., 1991, Nature 354:84-86), and combinatorialchemistry-derived molecular library made of D- and/or L-configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate directed phosphopeptide libraries; see,e.g., Songyang, Z. et. al., 1993, Cell 72:767-778).

5.5. Treatment and Prevention of Disorders Involving the ImidazolineSystem

Diseases and disorders involving imidazoline receptors are treated orprevented by administration of a compound that either promotes orantagonizes the activity stimulated by binding of IAA-RP, IAA-R or arelated congener to imidazoline receptors.

In specific embodiments, compositions that promote IAA-RP or IAA-Rfunction are administered to an individual: (1) in diseases or disordersinvolving an absence or decreased (relative to normal or desired) levelof IAA-RP or IAA-R, for example, in patients where the IAA-RP or IAA-Rare lacking, biologically inactive or underactive, or under expressed;or (2) in diseases or disorders wherein in vitro (or in vivo) assaysindicate that the utility of IAA-RP, IAA-R or a related congener agonistadministration. The absence or decreased level of IAA-RP or IAA-R can bereadily detected, e.g., by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying levels. Many methods standard in the art canbe thus employed, including but not limited to assays for biologicalactivity (e.g. arachidonic acid, insulin, and catecholamines release),immunoassays to detect and/or visualize IAA-RP or IAA-R (e.g., Westernblot, immunoprecipitation followed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, immunocytochemistry, ELISA assays,etc.).

In some instances, imidazoline system-based disorders can be treated orprevented by administration of a composition that antagonizes (inhibits)IAA-RP or IAA-R function. Compounds that inhibit IAA-RP or IAA-Rfunction can be identified by use of known convenient in vitro assays,e.g., based on their ability to inhibit binding of IAA-RP or IAA-R toimidazoline receptors. Preferably, suitable in vitro or in vivo assaysare utilized to determine the effect of a specific compound and whetherits administration is indicated for treatment of the affected tissue.

In specific embodiments, compounds that inhibit IAA-RP or IAA-R functionare administered therapeutically (including prophylactically): (1) indiseases or disorders involving an increased (relative to normal ordesired) level of IAA-RP or IAA-R; or (2) in diseases or disorderswherein in vitro (or in vivo) assays indicate the utility of IAA-RP orIAA-R antagonist administration. The increased levels in IAA-RP or IAA-Rconcentration and/or function can be readily detected, e.g., byquantifying levels, by obtaining a patient tissue sample (e.g., frombiopsy tissue) and assaying it in vitro for levels of the molecules,and/or activity of the molecules. Many methods standard in the art canbe thus employed, including but not limited to assays for detectingrelease of arachidonic acid (e.g. in the case of disorders affected byimidazoine I₁ receptors), immunoassays to detect and/or visualize IAA-RPor IAA-R (e.g., immunocytochemistry, in situ hybridization, ELISAassays, etc.).

The compositions of the invention are preferably tested in vitro, andthen in vivo for the desired therapeutic or prophylactic activity, priorto use in humans. For example, in vitro assays can be used to determinewhether administration of a specific compound is indicated, including invitro cell culture assays in which a patient's tissue sample is grown inculture and exposed to the compound, and the effect of such compoundupon the tissue sample is observed. For example, compositions can betested for their ability to either stimulate or inhibit the binding ofIAA-RP, IAA-R or related congeners to imidazoline receptors.Alternatively, the ability of a compound to inhibit or stimulatereceptor mediated signal transduction events, such as release ofarachinoic acid (in the case of I₁ receptors) can be tested.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to rats,mice, chicken, cows, monkeys, rabbits, etc. For in vivo testing, priorto administration to humans, any animal model system known in the artmay be used.

5.6. Administration of Pharmaceutical Compositions

Various delivery systems are known and can be used to administer thecompositions of the invention, e.g. encapsulation in liposomes,microparticles, microcapsules. Methods of introduction include, but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The compounds maybe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. Pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent.

The amount of the compositions of the invention which may be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. However, suitable dosage ranges forintravenous administration are generally about 20-500 micrograms ofactive compound per kilogram body weight. Suitable dosage ranges forintranasal administration are generally about 0.01 pg/kg body weight to1 mg/kg body weight. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

EXAMPLE IAA-RP and IAA-R Bind to the Imidazoline Receptor

In the example detailed below, binding of IAA-RP, IAA-R and relatedcongeners to imidazoline receptors was demonstrated. In addition, IAA-RPinduced the release of arachidonic acid, an imidazoline I₁ receptormediated response in cultured medullary adrenal cells and the I₁receptor antagonist efaroxan was found to inhibit this IAA-RP-inducedrelease of arachidonic acid.

6.1. Materials and Methods 6.1.1. Tissue Preparation

Whole bovine brains and adrenal glands were obtained from a localslaughterhouse. Brains were immediately placed on a chilled glass plate,washed with ice-cold Krebs'-Henseleit buffer, and dissected. The rostralmedulla was isolated by transecting the brainstem rostrally at theposterior margin of the trapezoid body and caudally 1 cm caudal to theobex. The pia-arachnoid was removed, and the lateral medulla wasisolated by a sagittal section through the lateral margin of thepyramids, and then bisected. The ventral half was defined as theventrolateral medulla (VLM). Brain samples were transported on ice tothe laboratory and processed immediately.

Fresh bovine adrenal glands (<10 min post-mortem) were dissected free ofassociated fat and connective tissue and perfused retrogradely throughthe adrenal vein with 25 ml ice-cold Krebs'-Henseleit bicarbonatebuffer. The glands were drained and reperfused with Krebs'-Henseleitbuffer, drained, and then perfused again with 25 ml ice-coldKrebs'-Henseleit buffer containing 0.025% collagenase (type D, BoehingerMannheim). The glands were incubated with occasional mixing duringtransport to the laboratory (about one hour), then perfused with 25 mlfresh buffer containing collagenase and incubated for 30 min at 35° C.The digested glands were split and the medulla was scraped away from thecortex and placed under 20 ml of buffer containing collagenase. Adrenalmedullae were mechanically minced (Tekmar Tissuemincer, setting 40 for30 sec) and incubated with stirring for 30 min at 37° C. The digest wasfiltered through stainless steel mesh and the filtrate centrifuged at200×g for 10 min at 20° C. The cell pellet was resuspended in 30 mlKrebs' without collagenase, recentrifuged, lysed by flash-freezing, andstored overnight at −70° C. About 70% of the cells isolated in this wayare chromaffin cells as shown by neutral red staining.

6.1.2 Membrane Preparation

Fresh bovine VLM was homogenized by using a polytron (TekmarTissuemizer, setting 80 for 2×15 sec) in 20 volumes of ice-coldHepes-buffered isotonic sucrose (pH brought to 7.4 with Tris base)containing the protease inhibitors (1,10)-phenanthroline (100 μM) andphenylmethylsulfonyl fluoride (50 μM) in order to inhibit degradation ofreceptor protein. Bovine adrenomedullary cells were homogenized in 15 mlHepes-buffered isotonic sucrose by 10 strokes in a glass-glass hand-heldhomogenizer. All three homogenates were centrifuged at 1000×g for 5 minat 4° C. to remove nuclei and debris. The pellets (P1) were resuspendedin 20 ml of homogenization buffer, and centrifuged again at 1000×g for 5min. The combined supernatants were centrifuged at 48,000 g for 18 minat 4° C., and the resulting P2 pellet was resuspended in 10 to 25volumes of 50 mM Tris-HCl buffer (pH 7.7) containing 5 mM EDTA. Afterrecentrifugation at 48,000 g for 18 min, the resulting membrane pelletwas resuspended in Tris-HCl containing 25 mM NaCl, preincubated for 30min at 25° C., chilled on ice, centrifuged again, resuspended a finaltime in Tris-HCl alone, centrifuged, flash-frozen, and stored at −70° C.for up to three months.

6.1.3. [³H]Clonidine and [¹²⁵I]p-Iodoclonidine Binding Assays

Radioligand binding assays with [³H]clonidine or [¹²³I]p-iodoclonidinefor determination of specific membrane binding to VLM and renal medullaimidazoline sites and α₂-adrenergic receptors were performed by amodification of methods previously described (Ernsberger P. et al.,1997, J. Hypertension 1997, 15:S9-S23). Membranes were slowly thawed andresuspended in Tris-Hepes buffer (5.0 mM; pH 7.7 at 25° C., containing0.5 mM EDTA, 0.5 mM EGTA, and 0.5 mM MgCl₂) at a concentration of 1 mgprotein/ml for the VLM, 4 mg protein/ml for the renal medulla, and 0.2mg protein/ml for adrenomedullary cells. Assays were conducted in atotal volume of 250 μl in polypropylene 96-well plates (BeckmanMacrowell), and each well contained 125 μl membrane suspension, 25 μlradioligand, and 100 μl drug or vehicle. Incubations were initiated bythe addition of membrane and were carried out for 30 min at 22° C.Nonspecific binding was defined in the presence of 10 μM BDF-6143, animidazoline adrenergic agent. Specific μ₂-adrenergic binding was definedby inhibition with (−) epinephrine (0.1 mM). In experiments usingcatecholamines, all samples contained ascorbic acid in a finalconcentration of 0.001%. Incubations were terminated by vacuumfiltration using a cell harvester (Brandel) equipped with Teflon tubingto reduce absorption of the radioligand over glass fiber filters(Schleicher & Schuell #34) which were preincubated for 4 h at 4° C. in0.03% polyethylimine to reduce nonspecific binding to the filter. Thefilters were washed four times with 5 ml ice-cold Tris-HCl, placed inscintillation vials, covered with 4 ml scintillation cocktail (BioSafeII, Research Products International), and counted at 50% efficiency(Beckman LS5801). Protein was assayed by the bicinchoninic acid method.

Data were obtained as dpm and transferred to the Equilibrium BindingData Analysis (EBDA) program for initial processing; 4 to 10 experimentswere analyzed simultaneously by using the LIGAND program for nonlinearcurve-fitting. Protein assay data were also analyzed by nonlinearcurve-fitting.

[³H]Clonidine (60-80 Ci/mmol) and [¹²⁵I]p-iodoclonidine (2200 Ci/mmol)were obtained from New England Nuclear (Boston, Mass.), stored at −20°C. in ethanol and diluted in water prior to assay. Stock solutions ofboth compounds were made in 0.01 M acetic acid up to one week prior touse. Epinephrine and clonidine were purchased from Sigma Chemical (St.Louis, Mo.). Cimetidine was purchased from Research BiochemicalsInternational (Natick, Mass.).

Assays for measuring imidazoline receptor-mediated arachidonic acidrelease from PC12 cells is as described in Ernsberger et al., (1995,FASEB J. 9:A114).

6.1.4. Preparation of Antibodies

IAA-RP linked to KLH was prepared by first reacting disodium IAA-RP(11.1 μmol) with 10 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimideHCl (EDCI; Sigma) in 0.5 mL of acidified saline (pH 6). After 5 min thiswas mixed with freshly dialyzed KLH protein (0.5 mg in 0.5 ml of 10 mMphosphate buffered saline [PBS; pH 7.4]). The solution was incubated atroom temperature for 5-7 h, then dialyzed repeatedly against PBS at 4°C. Five male mice were injected (approx. 0.2 ml i.p.) with 200-250 μgKLH linked to IAA-RP prepared in Freund's Complete Adjuvant in oil. Eachmouse received booster injections (i.v.) of KLH-IAA-RP in PBS after 6weeks and 18 weeks. Five days after the last injection, the mouse withhighest IAA-RP antibody titer was anesthesized with ether, then bled.Whole blood was collected, allowed to clot, then centrifuged (3000 g).The untreated plasma was the source of polyclonal antibodies againstIAA-RP (pAb-IAA-RP). The mouse was euthanized by cervical dislocationthen underwent spleenectomy, for preparation of monoclonal antibodies.

IAA-RP (as well as IAA-R and numerous potential congeners and relatedimidazoles) was similarly linked to BSA (Sigma) as was done for KLHabove. After dissolving in 0.15 M NaHCO₃, the solution was applied(approx. 0.5 μg/well) to 96 well plastic Maxisorp immunoplates (Nunc)for use in an Enzyme-Linked Immunosorbent Assay (ELISA) method.Solutions (100 μl) of pAb-IAA-RP (diluted up to 1:8000 in PBS) appliedto treated ELISA plates containing bound BSA-IAA-RP were incubated for60 min at 37° C. After washing (5 times with PBS), 100 μl of a secondantibody (goat-antimouse; 1:1000 dilution in PBS/1% BSA) linked toperoxidase (Kirkegaard and Perry Labs, Inc.) was added to each well andincubated for 60 min at 37° C. After washing (5 times with PBS), 95 μlof peroxidase substrate solution, ABTS (Kirkegaard and Perry Labs,Inc.), was added to each well. Plates were incubated for 20 min-4h at37° C. Optical density was assayed at 414 nm using a Spectra Max UV/Vspectrometer as the detector system.

6.2. Results

The data presented in FIG. 2, and Table 1 below, demonstrate that IAA-RPinhibits binding of labeled clonidine to bovine RVLM membranes. TABLE 1Binding parameters for IAA-RP inhibition of [¹²⁵I]p-iodoclonidinebinding to bovine RVLM membranes Imidazoline I Sites α₂-Adrenergic SitesCondition K_(i) (nM) Percent Sites K_(i) (μM) Percent Sites Control 160± 38 71 ± 5 57 ± 33 29 ± 5 With epinephrine 100 ± 19 86 ± 4 60 ± 48 14 ±4 With cimetidine  0 ± 0 210 ± 32  100 ± 10 K_(i) values are IAA-RP concentration in nM (imidazoline sites) or μM(α₂-adrenergic sites)± the standard error of the estimate and wereobtained by nonlinear curve-fitting to a two-component logisticequation. IAA-RP distinguished two populations of [¹²⁵I]p-iodoclonidinebinding sites. Sites with a high affinity for IAA-RP represented 71% ofthe total sites in the control condition, increased to 86% afterselective masking of α₂-adrenergic receptors with epinephrine and wereeliminated by the addition of cimetidine to mask imidazoline sites.Conversely, sites with a low affinity for IAA-RP were diminished in thepresence of epinephrine, but were predominant in the presence ofcimetidine. These data indicate that VLM imidazoline receptor sites havea high affinity for IAA-RP whereas α₂-adrenergic receptors have lowaffinity.

Authentic IAA-RP and IAA-R were both able to displace [¹²⁵I-]p-iodoclonidine; IAA-RP had a 3-30 fold greater affinity than IAA-R. Inaddition, incubation of brain-derived CDS with exogenous phosphatasereduced the binding affinity of CDS to adrenomedullary cell membranes(FIG. 3). In addition, as demonstrated in FIG. 4, CDS activity derivedfrom bovine brain extracts and synthetic IAA-RP, each stimulated releaseof arachidonic acid from PC-12 cells. This effect was blocked by theselective imidazoline I₁ antagonist, efaroxan. In addition, CDS activitywas shown to stimulate the release of catecholamines from adrenalchromaffin cells. In experiments using insulinoma cells in culture(e.g., β-TC3 cells), it was also observed that IAA-RP was capable ofstimulating insulin release indicating that such cells are responsive toIAA-RP.

EXAMPLE Stimulation of Insulin Release from Pancreatic Cells by IAA-RPand IAA-R

The example described below demonstrates that IAA-RP (tele-linked)stimulated release of insulin from cultured insulinoma cells (β-TC3cells), further indicating that IAA-RP is a CDS compound. Furthermore,it suggests a useful model system for assaying imidazoline inducedchanges in K⁺ channel activity such as, for example, activation (i.e.,closure) of K⁺ ATP-sensitive channels and/or regulation of transmitterrelease in regions of the body other than the pancreas. Such assaysprovide useful model systems for studying the relationship betweenimidazolines, neuropsychiatric and neurodegenerative disorders, and cellpathology and death associated with potassium channel dysfunction canoccur with K⁺ ATP channels.

7.1. Materials and Methods

Islets of Langerhans from male Wistar rats were isolated by collagenasedigestion in a medium of bicarbonate-buffered physiological salinesolution containing 4 mM D-glucose and 1 mM CaCl₂. Islets were selectedunder a binocular dissecting microscope and were used within two hoursof isolation. Islets from humans were isolated from heart-beatingcadaver organ donors by collagenase digestion and density gradientcentrifugation (Chan, S. L. F., et al., 1997, Brit. J. Pharmacol.120:926-932).

Incubations were done in 96-well plates. Isolated islets were incubatedin 100 μl buffer solution supplemented with bovine serum albumin inhumidified air: CO₂ (95:5%) at 37° C. in the presence of test agents. Toeliminate any potential alpha-2 responses, yohimbine (10 μM), a potentalpha2-adrenergic blocker, was included in all incubations. After 60 minincubation, samples of medium were removed for measurement of insulinrelease by radioimmunoassay using anti-bovine insulin antiserum.Presence of insulin in the test media were compared to controls;positive differences represented amounts of insulin released. Inreversal experiments, diazoxide (e.g., 200 μM) was pre-incubated toblock glucose-induced insulin release. In these studies, test reagentswere used to determine if they were capable of overcoming diazoxide'sinhibitory effects on K⁺ ATP channels, a well known action ofimidazoline secretogogues in islet cells. For example, efaraxan (aninhibitor of I-1 responses in adrenal medullary cells) is a well knownI-3 agonist, i.e., it stimulates (at 100 μM) release of insulin andovercomes diazoxide inhibition thus illustrating again the dictomybetween I₁ and I₃ receptor subtypes.

7.2 Results

The tele-isomers of IAA-RP and IAA-R (FIG. 1) and some of its congenerswere evaluated in isolated pancreatic beta-cells harvested from normalrats and humans after autopsy. In direct stimulation experiments inrats, the tele-isomers of IAA-RP and IAA-R were potent stimulants ofinsulin release; the magnitude of response was dose-dependent,oftentimes effective at concentrations as low as 10 nM. The pros-isomerof IAA-R likewise stimulated insulin release with effects similar to oreven greater than that seen with tele IAA-RP. Furthermore, tele-linkedIAA-RP and tele- and pros-IAA-R were each able to reverse the inhibitoryaction of diazoxide on glucose-induced insulin release.

Diazoxide opens ATP-sensitive K⁺ channels; a common feature ofimidazolines active at K⁺ channels (in particular, pancreatic I₃receptors) is that such imidazolines reverse effects of diazoxide(Morgan, N. G., et al., 1995, Ann. N.Y. Acad. Sci. 763: 361-373; Chan,S. L. F., et al., 1997, Brit. J. Pharmacol. 120: 926-932).

As demonstrated, the imidazoleacetic acid-linked ribotides and ribosidesare potent stimulants at yet another group of imidazoline receptors, theI₃ subtypes. The I₃ subtypes are associated with K⁺ ATP-sensitivechannels, and the latter are present in many tissues of the body,particularly in the brain. Since IAA-RP immunoreactive cells areparticularly rich in selected regions of the brain (e.g., particularlyin the RVLM region of the brainstem), it is likely that theimidazole-linked ribotides and ribosides and their congeners also affectK⁺ channels, for example K⁺-ATP-sensitive channels, present in nervoustissue. Such localization in the VLM again confirms that IAA-RP is aCDS.

The present invention is not to be limit in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A composition comprising imidazoleacetic acid-ribotide (IAA-RP) and apharmaceutically acceptable carrier.
 2. A composition comprisingimidazoleacetic acid-riboside (IAA-R) and a pharmaceutically acceptablecarrier.
 3. A composition comprising one or mor congeners ofimidazoleacetic acid-ribotide or imidazoleacetic acid-riboside and apharmaceutically acceptable carrier.
 4. (Cancelled)
 5. (Cancelled) 6.(Cancelled)
 7. An antibody which is capable of binding to animidazoleacetic acid-ribotide or an imidazoleacetic acid-riboside.
 8. Amethod of diagnosing a disease or disorder characterized by an aberrantlevel of imidazoleacetic acid-riboside or imidazoleacetic acid-ribotidein a subject, comprising measuring the level of imidazoleaceticacid-riboside or imidazoleacetic acid-ribotide in a sample derived fromthe subject, in which an increase or decrease in the level ofimidazoleacetic acid-riboside or imidazoleacetic acid-ribotide, relativeto the level of imidazoleacetic acid-riboside or imidazoleaceticacid-ribotide found in an analogous sample derived from a subject nothaving the disease indicates the presence of the disease or disorder. 9.The method of claim 8 wherein the disease or disorder is hypertension.10. The method of claim 8 wherein the disease or disorder is apsychiatric or neurological disorder.
 11. The method of claim 8 whereinthe disorder is a pancreatic disorder.
 12. A method for assaying forcompounds that modulate the activity of imidazoleacetic acid-riboside orimidazoleacetic acid-ribotide comprising: (a) contacting a cell whichexpresses imidazoline receptors with imidazoleacetic acid-riboside orimidazoleacetic acid-ribotide in the presence of a test compound or avehicle control; and (b) determining whether the test compound decreasesthe level of imidazoline receptor activation as compared to those cellscontacted with a vehicle control.
 13. The method of claim 12, whereinactivation of the imidazoline receptor is assayed by measuring the levelof arachidonic acid release.
 14. The method of claim 12, whereinactivation of the imidazoline receptor is assayed by measuring the levelof insulin secretion.
 15. The method of claim 12, wherein the activationof the imidazoline receptor is assayed by measuring the activation ofK+/ATP channels.
 16. A method for identifying compounds that bind to animidazoleacetic acid-ribotide or imidazoleacetic acid-ribosidecomprising: (a) preparing a reaction mixture comprising animidazoleacetic acid-ribotide or imidazoleacetic acid-riboside and atest compound under conditions and for time sufficient to allow thecomponents of the mixture to interact and bind, (b) identifying thebound test compound.